Human immune therapies using a CD27 agonist in combination with another immune agonist to treat cancer

ABSTRACT

Methods of inducing T cell proliferation and expansion in vivo for treating conditions wherein antigen-specific T cell immune response are therapeutically desirable such as cancer, infection, inflammation, allergy and autoimmunity and for enhancing the efficacy of vaccines are provided. These methods comprise the administration of at least one CD27 agonist, preferably an agonistic CD27 antibody, alone or in association with another moiety such as immune stimulant or immune modulator such as an anti-CD40, OX-40, 4-1BB, or CTLA-4 antibody or an agent that depletes regulatory cells, or a cytokine. These mono and combination therapies may also optionally include the administration of a desired antigen such as a tumor antigen, an allergen, an autoantigen, or an antigen specific to an infectious agent or pathogen against which a T cell response (often CD8+) is desirably elicited.

FIELD OF THE INVENTION

The invention generally relates to the use of a CD27 agonist, preferablya CD27 agonistic antibody, as an adjuvant for promoting T-cell immunityin a subject in need thereof, e.g., a subject with cancer, infection,autoimmune disease, allergy or inflammatory disorder or a subject beingadministered a vaccine. Most preferably the CD27 agonist will be anintact human, humanized, or chimeric antibody or a fragment thereof ormay comprise a single chain antibody such as an scFv that specificallybinds human CD27. Alternatively, the agonist may comprise an engineeredantibody which has been modified to enhance or diminish its interactionswith host effector systems or to reduce adverse side effects. In somepreferred embodiments the binding of these agonistic anti-CD70antibodies to immune cells will not be blocked by CD70 or relatedligands.

Additionally, the invention provides novel adjuvant combinations,preferably synergistic combinations, for eliciting an enhanced T cellimmune response in a subject in need thereof comprising (i) at least oneCD27 agonist and (ii) another immunostimulant or immunomodulator, e.g. aCD40 antibody, CD28 antibody, OX40 antibody, 4-1BB antibody,anti-CTLA-4, TLR (toll like receptor) agonist or a moiety that depletesregulatory T cells, or a cytokine such as an interleukin e.g., IL-2 oran interferon (beta, beta, gamma, et al.). Similarly, these adjuvantcombinations are useful in treating conditions wherein enhanced T cellimmunity is therapeutically desired such as cancer, infectious diseases,allergy, autoimmunity, inflammatory disorders and for enhancing theefficacy of vaccines.

The subject monotherapies involving the administration of a CD27agonist. preferably an agonistic CD27 antibody the binding of which toimmune cells is preferably is not blocked by CD70 and combinationtherapies involving the administration of a CD27 agonist and anothermoiety such as another immune modulator or therapeutic agent are usefulin enhancing CTL immunity and T cell proliferation and survival in vivo.As discussed in greater detail infra, this invention is based at leastin part on the inventors' surprising observation that CD27: CD70interactions are required for the agonistic activity of CD40 antibodies(anti-CD70 mAb completely blocks the activity of agonistic CD40 showingthat CD27:CD70 interactions are required ‘downstream’ of CD40triggering). This observation suggested the therapeutic potential ofCD27 agonistic antibodies as therapeutic adjuvants and for activatingand inducing the expansion of antigen-specific T cells, particularlyCD8+ T cells i.e., effector and memory CD8+ effector cells. WhileApplicants do not want to be bound by their hypothesis, they theorizethat the agonistic property of the subject anti-CD27 antibodies on Tcells may be attributable, at least in part, to the fact that thebinding of such antibodies may not be blocked by CD70. However, theinvention is not limited thereto and embraces the use of any anti-CD27agonistic antibody or conjugate or fragment thereof that has anagonistic effect on T cell immunity. As described in detail infra, thepresent mono and combination therapies are especially useful in treatinghumans or other mammals with lymphomas and other cancers or infectiousconditions wherein enhanced antigen-specific CTL immune responses aretherapeutically desirable.

As noted the CD27 agonist may be administered alone or in conjunctionwith other therapeutic moieties such as other immune adjuvants orimmunostimulators or immune modulators, with or without a suitableantigen, such as a cancer peptide. For example, in one embodiment theCD27 agonist may be administered in association with a CD40 agonisticantibody. In this embodiment of the invention the CD40 agonist maycomprise a chimeric agonistic anti-human CD40 antibody referred toherein as LOB 7/4 or a humanized variant thereof. This chimeric antibodyhas been demonstrated by the inventors to elicit potent anti-tumoreffects on a range of CD40 expressing tumors and to potentiate cellularimmunity. Based on these results the use of this chimeric antibody andvariants thereof, e.g., human or humanized versions thereof as an immuneadjuvant or therapeutic for treating various chronic diseases includingcancers (CD40 positive and negative), especially solid tumors as well asits use as an immune adjuvant for treating infectious diseases,autoimmune diseases, allergic and inflammatory diseases are taught in anearlier patent application by some of the inventors herein.

In preferred embodiments of the invention the subject CD27 agonist isused to treat cancer, infectious diseases, autoimmune disorders,allergic disorders, or inflammatory disorders alone or with an antigenor is administered in conjunction with a vaccine or other therapeuticagents or immunotherapeutic, e.g., an antibody to CD40, OX40, CD28,CTLA-4, or 4-1BB; a TLR agonist; a moiety that depletes regulatory Tcells; a cytokine; an anti-angiogenesis agent; or a chemotherapeutic.Throughout this application when combination therapies are mentioned, itshould be understood that the respective moieties such as agonisticantibodies may be administered separately or in combination, e.g. in thesame or different compositions, and administration of the respectivemoieties may be effected in any order. Also, these combination andmonotherapies are intended to embrace the administration of additionalmoieties useful in treating the particular disease or condition.

BACKGROUND OF THE INVENTION

It is now widely recognized that the generation of protective immunitydepends not only on exposure to antigen, but also the context in whichthe antigen is encountered. Numerous examples exist in whichintroduction of a novel antigen into a host in a non-inflammatorycontext generates immunological tolerance rather than long-term immunitywhereas exposure to antigen in the presence of an inflammatory agent(adjuvant) induces immunity. (Mondino et al., Proc. Natl. Acad. Sci.,USA 93:2245 (1996); Pulendran et al., J. Exp. Med. 188:2075 (1998);Jenkins et al., Immunity 1:443 (1994); and Kearney et al., Immunity1:327 (1994)). Since it can mean the difference between tolerance andimmunity, much effort has gone into discovering the “adjuvants” presentwithin infectious agents that stimulate the molecular pathways involvedin creating the appropriate immunogenic context of antigen presentation.

CD27 is a member of the tumor necrosis factor receptor (TNFR) superfamily which also includes TNFR type I and II (CD120a and b), nervegrowth factor receptor (NGFR), CD30, Fas/Apo-1 (CD95), CD40, 4-1BB andOX40. These proteins are known to play key roles in cell growth,survival, and differentiation as well as apoptosis or programmed celldeath. Homology among these family members is restricted to theextracellular region and is characterized by the presence of a cysteineknot motif which occurs three times in CD27 (McDonald et al., Cell73:4121-424 (1993)).

CD27 is a glycosylated, type I transmembrane protein of about 55kilodaltons and exists as homodimers with a disulfide bridge linking thetwo monomers. The disulfide bridge is in the extracellular domain closeto the membrane (Camerini et al., J Immunol. 147:3165-69 (1991)). Theligand for CD27, CD70, belongs to the TNF family of ligands. CD70 is atype II transmembrane protein with an apparent molecular weight of 50 kd(Goodwin et al., Cell 73:447-456 (1993)). Based on homology to TNF alphaand beta, CD70 was predicted to have a trimeric structure made up ofthree identical subunits which interact with three CD27 homodimers(Peitsch et al., Mol. Immunol. 152:1756-1761 (1994)). TNF alpha whichalso is a type II transmembrane protein, is released from the cell byproteolytic cleavage, whereas TNF beta and NGF are secreted.

CD27 and its ligand CD70 are expressed on discrete populations of T andB cells. CD27 is expressed on resting T cells and CD70 on activated Tand B cells and dendritic cells. Within T cell subsets, CD27 is stablyexpressed on CD45RA+ cells even after activation, whereas on CD45RO+cells it is weakly expressed and lost after activation. (Sugita et al.,J Immunol. 149:3208-3216 (1992); Hintzen et al., J Immunol.151:2426-2435 (1993)). On CD45RA+ cells, activation by various meansresults in the upregulation of CD27 expression (Hintzen et al., JImmunol. 151:2426-2435 (1993)). CD27 is highly expressed on most of theB cell non-Hodgkin's lymphomas and B cell chronic lymphocytic leukemias.(Ranheim et al., Blood 85:3556-3565) The B cell lines Ramos and Raji,also express significant levels of CD27 and CD70.

Ligation of CD27 along with treatment of T cells with sub-optimal doseof PMA, PHA, anti-CD2 or anti-CD3 antibodies is also known to result inthe proliferation of T cells, thus defining a co-stimulatory role forCD27. It has also been reported that CD27-mediated co-stimulatoryeffects can be specifically inhibited using an anti-CD27 antibody orrecombinant soluble CD27 or anti-CD70 antibody and that ligation of CD27via its ligand, CD70 can generate cytolytic T cells. (Goodwin et al.,Cell 73:447-456 (1993)).

Another co-stimulatory molecule which is known to regulate adaptiveimmunity is CD40. CD40 is a member of the TNF receptor superfamily andis essential for a spectrum of cell-mediated immune responses andrequired for the development of T cell dependent humoral immunity(Aruffo et al., Cell 72:291 (1993); Farrington et al., Proc Natl AcadSci., USA 91:1099 (1994); Renshaw et al., J Exp Med 180:1889 (1994)). Inits natural role, CD40-ligand expressed on CD4+ T cells interacts withCD40 expressed on DCs or B cells, promoting increased activation of theAPC and, concomitantly, further activation of the T cell (Liu et alSemin Immunol 9:235 (1994); Bishop et al., Cytokine Growth Factor Rev14:297 (2003)). For DCs, CD40 ligation classically leads to a responsesimilar to stimulation through TLRs such as activation markerupregulation and inflammatory cytokine production (Quezada et al. AnnuRev Immunol 22:307 (2004); O'Sullivan B and Thomas R Crit Rev Immunol22:83 (2003)) Its importance in CD8 responses was demonstrated bystudies showing that stimulation of APCs through CD40 rescuedCD4-dependent CD8+ T cell responses in the absence of CD4 cells(Lefrancois et al., J Immunol. 164:725 (2000); Bennett et al., Nature393:478 (1998); Ridge et al., Nature 393:474 (1998); Schoenberger etal., Nature 393:474 (1998). This finding sparked much speculation thatCD40 agonists alone could potentially rescue failing CD8+ T cellresponses in some disease settings (French et al., Nature Medicine1999).

Other studies, however, have demonstrated that CD40 stimulation aloneinsufficiently promotes long-term immunity. In some model systems,anti-CD40 treatment alone insufficiently promoted long-term immunity,i.e., yields ineffective inflammatory cytokine production. as well asthe deletion of antigen-specific T cells (Mauri et al. Nat Med 6:673(2001); Kedl et al. Proc Natl Acad Sci., USA 98:10811 (2001)) andtermination of B cell responses (Erickson et al., J Clin Invest 109:613(2002)). Also, soluble trimerized CD40 ligand has been used in theclinic as an agonist for the CD40 pathway and what little has beenreported is consistent with the conclusion that stimulation of CD40alone fails to reconstitute all necessary signals for long term CD8+ Tcell immunity (Vonderheide et al., J Clin Oncol 19:3280 (2001)).

Both agonistic and antagonistic antibodies specific to CD40 have beensuggested to have potential as human therapeutics. Antagonisticanti-CD40 antibodies include those that (1) block CD40/CD40L interactionby at least 90% and have purported antineoplastic properties (Armitageet al., U.S. Pat. No. 5,674,492; Fanslow et al., 1995, Leukocyte TypingV Schlossman et al., eds., 1:555-556); (2) those that antagonizesignaling through CD40 (deBoer et al., U.S. Pat. No. 5,677,165) and (3)those that deliver a stimulatory signal through CD40 but do not increasethe interaction between CD40 and CD40L, e.g., G28-5, (Ledbetter et al.,U.S. Pat. No. 5,182,368; PCT WO 96/18413).

Agonistic anti-CD40 antibodies have been reported by several groups. Forexample, one mAb, CD40.4 (5C3) (PharMingen, San Diego, Calif.) has beenreported to increase the interaction between CD40 and CD40L byapproximately 30-40% (Schlossman et al., eds., Leukocyte Typing, 1995,1:547-556). Additionally, Seattle Genetics in U.S. Pat. No. 6,843,989allege to provide methods of treating cancer in humans using anti-humanCD40 antibodies. These antibodies are alleged to deliver a stimulatorysignal, to enhance the interaction between CD40 and CD40L by at least45% and to enhance CD40L-mediated stimulation and to possess in vivoneoplastic activity. The exemplified antibody disclosed in the SeattleGenetics patent was derived from S2C6, an agonistic anti-human CD40antibody previously shown to deliver strong growth-promoting signals toB lymphocytes (Paulie et al., 1989, J Immunol. 142:590-595).

However, notwithstanding these prior reports, improved methods and humantherapies using adjuvants that promote Th1 immunity and which enhancethe activation and expansion of antigen specific CD8+ T cells, i.e. CD8+effector and memory cells are needed. Particularly, improved methods oftreating human cancer and other diseases using therapeutic adjuvantswhich are safe and effective, i.e., which do not elicit undesired sideeffects but which elicit substantial therapeutic effects, e.g.,anti-tumor effects are needed. The present invention satisfies this needand provides other advantages as well.

SUMMARY OF THE INVENTION

This invention provides novel methods of human treatment using a CD27agonist such as an agonistic CD27 antibody alone or in conjunction withanother therapeutic agent, e.g. another immunostimulant orimmunomodulatory agent such as an agonistic CD40 antibody, solubleCD40L, a 4-1BB:4-1BBL agonist, an OX40 agonist, TLR agonist, a moietythat depletes regulatory T cells, or a cytokine such as an interleukinor an interferon. As noted above in preferred embodiments the binding ofthese CD27 agonistic antibodies to immune cells will not be blocked byCD70 as this may have a beneficial effect on the agonistic activity ofthese antibodies on T cells. These combinations would normally beemployed together with a vaccine in the form or protein, peptides,immunogenic cells, or DNA. The results infra show that blocking theco-stimulation of CD27 has a profound effect on anti-tumor immunityelicited by an agonistic anti-CD40 antibody much more than blocking4-1BB co-stimulation, and that this is apparently the result of a severeimpairment in CD8+ T cell expansion during CD27-CD70 blockade. Theseresults suggest that CD27 agonists, e.g., agonist anti-CD27 antibodiesmay be used as therapeutic adjuvants for promoting CD8+ T cell expansionand in treating conditions wherein this is therapeutically desirablesuch as cancer, infectious disorders, autoimmunity, allergic disorders,and inflammatory disorders. Also, it suggested that a CD27 agonist,e.g., a CD27 agonistic antibody may be administered in association withan antigen or vaccine in order to promote antigen-specific CD8+ T cellimmunity. In an especially preferred embodiment the CD27 agonist will beused to treat a cancer such as a lymphoma or other cancers identifiedinfra.

While the mechanism by which anti-CD40 monoclonal antibodies generateimmune responses against tumors in humans and rodents is not fullyunderstood, it appears to operate through the stimulation of dendriticcells to a level that boosts CTL responses and circumvents the need forCD4 helper cells. It is shown herein that agonistic CD40 monoclonalantibody promotes strong expression of 4-1BB on the expanding CD8 cells,together with a modest loss of CD27 as effector CTL are generated.Interestingly, while CD40 mAb treatment caused a profound activation ofdendritic cells in tumor-bearing mice, the expression of 4-1BBL andCD70, the respective ligands for 4-1BB and CD27, was relatively weak andtransient. Despite this lack of expression their involvement wasestablished by showing that mAb (AT113-2:anti-4-1BBL, andTAN1-6:anti-CD70), which blocked the interaction CD70 block in thetherapeutic efficacy of CD40 mAb treatment. Likewise they had noinfluence on the cytotoxic activity of tumor-specific CTL cells eitherin vitro or in vivo. Based thereon, the present inventors concluded that4-1BB:4-1BBL and particularly CD27:CD70 interactions are pivotal inexplaining the activity of CD40 agonistic antibodies and that theseantibodies act by triggering CD8 T cell proliferation and survival.Moreover, the inventors show for the first time herein that an agonisticanti-CD27 antibody is highly effective by itself in protectinglymphoma-bearing mice (A31 and BCL1) indicating that CTL can begenerated at a later stage than DC and that agonistic CD27 antibodiesmay be used to treat cancer and other diseases wherein enhanced cellularimmunity is necessary. Importantly, this anti-CD27 mAb did not protectimmunocompromised SCID mice from these same tumors, underlining the needfor a functional immune system (CD8 T cells) to provide this protection.

Therefore, this invention provides novel mono and combination therapiesfor promoting T cell immunity in a subject in need thereof, e.g., asubject with lymphoma comprising the administration of at least one CD27agonist and optionally another moiety such as a CD40 agonist. As notedabove, if a CD40 agonist is utilized in association with the subjectCD27 agonistic antibody, the CD40 agonist will preferably comprise ananti-human CD40 antibody e.g., a chimeric antibody referred to herein asLOB 7/4 or a derivative thereof, e.g., humanized antibodies or fragmentsthereof containing the variable heavy and light sequences or CDRsderived from the LOB 7/4 antibody. The present inventors have found thatthis chimeric antibody possesses advantageous properties when used as atherapeutic, e.g. for treatment of cancer, especially CD40 expressinglymphomas and solid tumors.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1C contain the results of an experiment which shows that TAN1-6(anti-CD70 antibody) and AT113-2 (anti-4-1BBL) block the 4-1BB andCD70/CD27 interactions respectively in vitro (FIGS. 1A and 1B) andduring in vivo (FIG. 1C) responses.

FIG. 2 contains the results of an experiment that shows the effect ofanti-4-1BBL (AT113-2) and anti-CD70 (TAN1-6) on the therapeutic activityof anti-CD40 in BCL1 mice.

FIGS. 3A and 3B contain the results of an experiment showing the effectof anti-4-1BBL and anti-CD70 on the accumulation of CD8+ T cells and theeradication of BCL1 tumor after anti-CD40 mAb therapy. FIGS. 3A and 3Balso show the effects of the 4-1BBL and anti-CD70 mAb on the growth ofBCL1 (FIG. 3A) and the CD8 T-cell response (FIG. 3B) in the spleen ofCD40 mAb treated mice.

FIGS. 4A and 4B contain the results of an experiment that shows thatanti-4-1BBL (AT113-2) and anti-CD70 (TAN1-6) do not affect the anti-CD40induced phenotypic changes (FIG. 4A) or changes in the number of splenicDCs (FIG. 4B) from BCL1 mice.

FIGS. 5A-5C contain the results of an experiment that shows thatanti-4-1BBL and anti-CD70 antibodies do not inhibit the effector stageof the response in vivo (FIGS. 5A and 5B) or in vitro (FIG. 5C).

FIG. 6 contains the results of an experiment that demonstrates thetherapeutic efficacy and potency of agonistic CD27 monoclonal antibodiesagainst two B cell lymphomas (BCL1 and A31).

FIG. 7 contains experiments that show that the anti-CD27 mAb elicits astronger response (higher and longer) in OT-I cells than the CD70ligand. The figure shows the expansion of Ag-specific (OT-1) cells inmice given Ag+ control Ab, Ag+ soluble recombinant CD70 or an agonisticCD27 antibody according to the invention. The results in the figure showsubstantial expansion of CD8+OT-1 with the agonistic CD27 mAb. (SeeExample 6)

FIGS. 8A and 8B show mouse CD27 transiently expressing cells labeledwith anti-mouse CD27 antibodies. As shown therein both the test anti-ratanti-mouse CD27 (AT124-1) and positive control hamster anti-mouse CD27(LG3A10) labeled the mouse transfected cells (FIGS. 8A and 8B,respectively). Neither antibody bound to non-transfected cells (data notshown).

FIGS. 9A-9H show blocking of CD70 and CD27 with the anti-CD27 mAb. Thisfigure shows that AT 124-1 binds to CD27 on the surface of activatedmouse T cells and that this binding is not inhibited by the interactionof CD27 with its natural ligand CD70 (FIGS. 9A and 9E). By contrast, thecomparison commercially available anti-CD27 antibody (LG3A10; anon-agonist) was shown to compete for binding with CD70 (FIGS. 9C and9G). Controls for the rat anti-mouse CD27 (AT124-1) and the hamsteranti-mouse CD27 (LG3A10) antibodies are shown in FIGS. 9B, 9F, 9D and9H, respectively.

DETAILED DESCRIPTION OF THE INVENTION

Interactions between members of the TNFR superfamily and their TNFfamily ligands play a critical role in providing co-stimulation atseveral stages during the development of an effective, antigen-specificCD8 T-cell response to pathogens and tumors. Early in the response, theligation of CD40 on dendritic cells (DC) by its ligand, CD154 (CD4OL),on activated Th cells, induces the activation, or licensing, of DCs andpotentiates their ability to present antigen to naïve CD8 T cells(Bennett, Carbone et al. 1998; Ridge, Di Rosa et al. 1998; Toes,Schoenberger et al. 1998). Anti-CD40 mAb or soluble CD40 ligand (CD4OL),which can substitute for Th cells for DC licensing, have been shown tohave therapeutic potential in a number of settings requiring T cellresponses, including vaccination, and treatment of tumors, (French, Chanet al. 1999; Tutt, O'Brien et al. 2002; van Mierlo, den Boer et al.2002; Murray, Lu et al. 2003) viruses and infections (Rolph and Kaufman2001; Murray, Lu et al. 2003). The CD40-induced activation of DC ischaracterized by an increase in their expression of adhesion andco-stimulatory molecules, including ICAM-1, B7.1, B7.2, CD70 and 4-1BBligand (4-1BBL) (Cella, Scheidegger et al. 1996; Diehl, van Mierlo etal. 2002; Tesselaar, Xiao et al. 2003)). While initial antigen-specificCTL activation/priming is dependent on the CD28:B7 engagement (Lenschow,Walunas et al. 1996; Carreno and Collins 2002)), subsequent expansionand survival of effector cells is controlled by a plethora of additionalco-stimulatory interactions. The precise role of each of these pathwaysin maintaining memory responses is currently an area of activeinvestigation. Two receptors which appear critical in maintaining CTLresponses are the TNFR-family members 4-1BB (CD137) (DeBenedette,Shahinian et al. 1997; Futagawa, Akiba et al. 2002) and CD27 (Tesselaar,Xiao et al. 2003), which interaction with their respective ligands,4-1BBL and CD70 expressed mainly on DC. For example, it is known that,while anti-CD40 mAb driven CTL responses are helper independent, theyremain dependent on both CD28:B7 and CD27:CD70 interactions ((Prilliman,Lemmens et al. 2002; Tutt, O'Brien et al. 2002; Taraban, Rowley et al.2004). Less information is currently known of the importance of 4-1BBduring anti-CD40 mAb driven CTL responses, but it is clear that T-helpercell-dependent priming of CTL is at least partially dependent on 4-1BBand is markedly compromised by 4-1BB blockade (Diehl, van Mierlo et al.2002).

The present inventors have previously demonstrated that anti-CD40 mAbstimulates a helper-independent CTL response against a number ofsyngeneic lymphomas, that successfully eradicates existing tumors, andleaves the mice resistant to rechallenge (French, Chan et al. 1999;Tutt, O'Brien et al. 2002). Early clinical trials with anti-CD40 mAbshows clinical success and patient benefit.

By contrast this invention relates to the role of CD27 as a therapeuticimmune adjuvant and for promoting CD8+ T cell immunity, e.g., inlymphoma subjects. In an Ova model system, the inventors have recentlyshown that costimulation via CD27 is essential for CD40 mAb-mediatedpriming of ovalbumin-specific CTLs. Although priming of endogenousovalbumin-specific T cells was not seen when CD70-CD27 interaction wasabrogated, the priming of ovalbumin-specific TCR transgenic T cells(OT-I) was detected, albeit at markedly reduced level. Furthermore,while OT-I T cells primed in the absence of CD27 signaling were able todifferentiate into cytotoxic T cells, their ability to mount a secondaryresponse was defective. In light of these findings we wished to addressthe role of CD27 costimulation in CD40 mAb-mediated immunotherapy oflymphoma and to compare that with the role of its close relative 4-1BB.The results of these studies which are provided in the examples infrashow that blocking co-stimulation via CD27 has a more effect on theanti-tumor response elicited by an agonistic antibody and this effect ismuch more pronounced than blocking 4-1BB co-stimulation. The inventorsfurther believe that this is due to a severe impairment in CD8+ T cellexpansion during CD27-CD70 blockade.

Based thereon, the present invention provides novel methods of humantherapy by administering an immunologically promoting (adjuvant) ortherapeutically effective amount of (i) at least one CD27 agonist and(ii) optionally another therapeutic moiety which may comprise anotherimmunomodulator or immunostimulant such as a CD40 agonist, an OX-40agonist, a 4-1BB agonist, anti-CTLA-4, a moiety that depletes regulatoryT cells (Treg), or a cytokine such as an interferon or interleukin ormay comprise a drug or chemotherapeutic agent. The CD27 agonist alone orin combination results in the potentiation of the increase in CD8+ Tcell proliferation and CTL immune responses, e.g., those elicited by anCD40 agonist such as an agonistic CD40 antibody.

The present invention further provides for the first time agonisticanti-CD27 antibodies useful for promoting T cell immunity. In apreferred embodiment the binding of these agonistic antibodies will notbe affected (inhibited) by CD70. While this is not essential to thepresent invention, it is believed that agonistic antibodies that do notcompete with CD70 may possess advantageous properties when used asimmune adjuvants, e.g., in the treatment of cancers such as lymphomas.

The CD27 agonist will preferably comprise an agonistic anti-CD27antibody. This antibody which may or may not compete with CD70 willpreferably comprise a human, humanized, chimeric agonistic anti-humanCD27 antibody that preferably comprises a human constant domain whichmay be an IgG1, IgG2, IgG3 IgG4, IgM, IgD, IgE, IgA1 or IgA2 humanconstant domain. These constant domains may be modified if desired inorder to enhance or modify effector function. Also, the antibody may bemutated to remove or alter glycosylation. Similarly, if anotheragonistic antibody is used along with the CD27 agonistic antibody theysimilarly will preferably be human, humanized or chimeric antibodiespreferably containing a human constant domain and may be mutated asdescribed above with respect to the CD27 agonistic antibody. Also,single chain antibodies and antibody fragments are within the scope ofthe invention such as Fabs, scFvs, minibodies and the like.

In an exemplary embodiment if a CD40 agonist is co-administered with theCD27 agonist the CD40 agonist will comprise a chimeric anti-human CD40antibody referred to herein as LOB 7/4, or a variant thereof, or afragment thereof, especially humanized versions thereof, and/orantibodies antibody fragments which possess the same epitopicspecificity as LOB 7/4 or which compete with LOB 7/4 for binding tohuman CD40.

In an exemplary embodiment the present invention provides novel methodsof treating human cancer, such as solid tumors and lymphomas byadministering a therapeutically effective amount of a CD27 agonist suchas an agonistic anti-CD27 antibody. Optionally, this agonist may beadministered in association with another agonist or cytokine, e.g., onewhich elicits a synergistic effect therewith such as a CD40 agonist, a4-1BB:4-1BBL agonist such as a 4-1BBL agonistic antibody, a 4-1BBagonist such as an agonistic 4-1BB antibody, anti-CTLA-4 or aninterleukin such as IL-2 or an interferon such as an alpha, beta, orgamma interferon or a moiety that results in Treg depletion. Theseimmune combination may be administered together or in combination. Theseagonists or agonist/cytokine combinations may be in the same compositionor different compositions. Preferably they are administered synchronousor close to synchronous to each other. Typically, these therapeuticmoieties are administered within 24 hours of each other, more typicallywithin 8 hours, and still more typically within 1-4 hours of each other.

Cancers treatable with the subject CD27 agonist, i.e., CD27 agonisticantibody include by way of example leukemia, acute lymphocytic leukemia,acute myelocytic leukemia, myeloblastic promyelocytic myelomonocyticmonocytic erythroleukemia, chronic leukemia, chronic myelocytic(granulocytic) leukemia, chronic lymphocytic leukemia, Polycythemia veraLymphoma, Hodgkin's disease, non-Hodgkin's disease, multiple myeloma,Waldenstrom's macroglobulinemia, heavy chain disease, solid tumors,sarcomas, and carcinomas, fibrosarcoma, myxosarcoma, liposarcoma,chrondrosarcoma, osteogenic sarcoma, osteosarcoma, chordoma,angiosarcoma, endotheliosarcoma, lymphangiosarcoma,lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor,leiomyosarcoma, rhabdomyosarcoma, colon sarcoma, colorectal carcinoma,pancreatic cancer, breast cancer, ovarian cancer, prostate cancer,squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweatgland carcinoma, sebaceous gland carcinoma, papillary carcinoma,papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma,bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile ductcarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor,cervical cancer, uterine cancer, testicular tumor, lung carcinoma, smallcell lung carcinoma, non small cell lung carcinoma, bladder carcinoma,epithelial carcinoma, glioma, astrocytoma, medulloblastoma,craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acousticneuroma, oligodendroglioma, menangioma, melanoma, neuroblastoma,retinoblastoma, nasopharyngeal carcinoma, and esophageal carcinoma. Inpreferred embodiments the subject antibody is used to treat CD40expressing solid tumors such as CD40 expressing melanoma, non-small lungcarcinoma, invasive duct breast carcinoma, diffuse large B celllymphoma, and other solid tumors which express CD40.

Still further the invention provides novel methods of potentiatingcellular immunity in a human subject in need of such treatment byadministering an amount of the subject adjuvant agonist combination to apatient in need thereof alone or in combination with another activeagent such as a cytokine and optionally an antigen.

Additionally, the present invention is directed to treating humaninflammatory diseases and deficiencies using the subject adjuvantcombination alone or in conjunction with other immune- andnon-immune-based therapeutics. Such conditions include by way of examplesystemic lupus erythematosus (SLE), scleroderma (e.g., CRST syndrome),inflammatory myositis, Sjogren's syndrome (SS), mixed connective tissuedisease (e.g., MCTD, Sharp's syndrome), rheumatoid arthritis, multiplesclerosis, inflammatory bowel disease (e.g., ulcerative colitis, Crohn'sdisease) acute respiratory distress syndrome, pulmonary inflamation,osteoporosis, delayed type sensitivity, asthma, primary biliarycirrhosis (PBC), and idiopathic thromboctytopenic purpura (ITP).

The subject methods which preferably administer an agonistic anti-humanCD27 antibody and optionally another immune stimulant or immunemodulator such as an agonistic CD40, OX-40, 4-1BB, or CTLA-4 antibody,moiety that depletes regulatory T cells or a cytokine will beadministered to a host in need of such treatment in order to elicit anenhanced antigen specific antitumor or cellular immune response. Inpreferred embodiments these antibodies will be administered to a subjecthaving or at risk of developing a cancer, an infection, particularly achronic infectious diseases e.g., involving a virus, bacteria orparasite; or an autoimmune, inflammatory or allergic condition. Forexample the subject antibody or combination can be used to elicitantigen specific cellular immune responses against HIV. HIV is a wellrecognized example of a disease wherein protective immunity almostcertainly will require the generation of potent and long-lived cellularimmune responses against the virus.

As noted, this invention provides agonistic antibody therapies andcombination therapies which can be used in the treatment of chronicinfectious diseases involving viruses, bacteria, fungi or parasites aswell as proliferative diseases such as cancer, autoimmune diseases,allergic disorders, and inflammatory diseases where effective treatmentrequires the elicitation of a potent cellular antigen specific immuneresponse.

The subject CD27 agonist may optionally be administered in combinationwith other immune adjuvants such as lymphokines and cytokines. Examplesthereof include interferons such as alpha, beta, and gamma interferon,interleukins such as IL-2, 11-4, IL-6, IL-13 et al., colony stimulatingfactors, TNFs, and the like.

Additionally, the subject anti-human CD40 antibodies may be administeredin combination with other antitumor agents such as chemotherapeutics andcytotoxins commonly used for treating cancer, agents that inhibitangiogenesis, and the like. These additional therapeutic agents may beadministered separately or in combination with the subject agonisticanti-CD27 antibody. Also, in some embodiments an effector moiety such asa chemotherapeutic may be directly or indirectly attached to the subjectanti-human CD27 or other optionally included agonistic antibody such asanti-human CD40, anti-OX-40, anti-4-1BB, anti-CTLA-4 etc. e.g., by theuse of a linker.

Further, in some embodiments the subject anti-human CD27 antibody ortherapeutic combination containing an agonistic CD27 antibody may beadministered in combination with a desired antigen or attached to anantigen.

Exemplary antigens include but are not limited to bacterial, viral,parasitic, allergens, autoantigens and tumor associated antigens. If aDNA based vaccine is used the antigen will be encoded by a sequence theadministered DNA construct. Alternatively, if the antigen isadministered as a conjugate the antigen will be a protein comprised inthe administered conjugate. Still further, the antigen is administeredseparately from the CD27 antibody and the antigen can take any form.Particularly, the antigen can include protein antigens, peptides, wholeinactivated organisms, and the like.

Specific examples of antigens that can be used in the invention includeantigens from hepatits A, B, C or D, influenza virus, Listeria,Clostridium botulinum, tuberculosis, tularemia, Variola major(smallpox), viral hemorrhagic fevers, Yersinia pestis (plague), HIV,herpes, pappilloma virus, and other antigens associated with infectiousagents. Other antigens include antigens associated with a tumor cell,antigens associated with autoimmune conditions, allergy and asthma.Administration of such an antigen in conjunction with the subjectagonistic anti-CD27 antibody can be used in a therapeutic orprophylactic vaccine for conferring immunity against such diseaseconditions.

In some embodiments the methods and compositions can be used to treat anindividual at risk of having an infection or has an infection byincluding an antigen from the infectious agent. An infection refers to adisease or condition attributable to the presence in the host of aforeign organism or an agent which reproduce within the host. A subjectat risk of having an infection is a subject that is predisposed todevelop an infection. Such an individual can include for example asubject with a known or suspected exposure to an infectious organism oragent. A subject at risk of having an infection can also include asubject with a condition associated with impaired ability to mount animmune response to an infectious agent or organism, for example asubject with a congenital or acquired immunodeficiency, an infant, anelderly person, a subject undergoing radiation or chemotherapy, asubject with a burn injury, a subject with a traumatic injury, a subjectundergoing surgery, or other invasive medical or dental procedure, orother immunocompromised individual.

Infections which may be treated or prevented using the subject agonisticantibody combinations potentially in combination with other immunepotentiators include bacterial, viral, fungal, and parasitic infections.Other less common types of infections also include are rickettsiae,mycoplasms, and agents causing scrapie, bovine spongiform encephalopathy(BSE), and prion diseases (for example kuru and Creutzfeldt-Jacobdisease). Examples of bacteria, viruses, fungi, and parasites thatinfect humans are well known. An infection may be acute, subacute,chronic or latent and it may be localized or systemic. Furthermore, theinfection can be predominantly intracellular or extracellular during atleast one phase of the infectious organism's agent's life cycle in thehost.

Bacterial infections against which the subject antibodies may be used topotentiate a cellular immune response include both Gram negative andGram positive bacteria. Examples of Gram positive bacteria include butare not limited to Pasteurella species, Staphylococci species, andStreptococci species. Examples of Gram negative bacteria include but arenot limited to Escherichia coli, Pseudomonas species, and Salmonellaspecies. Specific examples of infectious bacteria include but are notlimited to Heliobacter pyloris, Borrelia burgdorferi, Legionellapneumophilia, Mycobacteria spp. (for example M. tuberculosis, M. avium,M. intracellilare, M. kansaii, M. gordonae), Staphylococcus aureus,Neisseria gonorrhoeae, Neisseria meningitidis, Listeria monocytogeners,Streptococcus pyogenes, (group A Streptococcus), Streptococcusagalactiae (Group B Streptococcus), Streptococcus (viridans group),Streptococcus faecalis, streptococcus bovis, Streptococcus (aenorobicspp.), Streptococcus pneumoniae, pathogenic Campylobacter spp.,Enterococcus spp., Haemophilus influenzae, Bacillus anthracis,Corynebacterium diptheriae, Corynebacterium spp., Erysipelothrixrhusiopathie, Clostridium perfringens, Clostridium tetani, Enterobacteraerogenes, Klebsiella pneumoniae, Pasteurella multocida, Bacteroidesspp., Fusobacterium nucleatum, Streptobacillus moniliformis, Treponemapallidum, Treponema pertenue, Leptospira, Rickettsia, and Actinomycesisraelii.

Examples of viruses that cause infections in humans include but are notlimited to Retroviridae (for example human deficiency viruses, such asHIV-1 (also referred to as HTLV-III), HIV-II, LAC or IDLY-III/LAV orHIV-III and other isolates such as HIV-LP, Picornaviridae (for examplepoliovirus, hepatitis A, enteroviruses, human Coxsackie viruses,rhinoviruses, echoviruses), Calciviridae (for example strains that causegastroenteritis), Togaviridae (for example equine encephalitis viruses,rubella viruses), Flaviviridae (for example dengue viruses, encephalitisviruses, yellow fever viruses) Coronaviridae (for examplecoronaviruses), Rhabdoviridae (for example vesicular stomata viruses,rabies viruses), Filoviridae (for example Ebola viruses) Paramyxoviridae(for example parainfluenza viruses, mumps viruses, measles virus,respiratory syncytial virus), Orthomyxoviridae (for example influenzaviruses), Bungaviridae (for example Hataan viruses, bunga viruses,phleoboviruses, and Nairo viruses), Arena viridae (hemorrhagic feverviruses), Reoviridae (for example reoviruses, orbiviruses, rotaviruses),Bimaviridae, Hepadnaviridae (hepatitis B virus), Parvoviridae(parvoviruses), Papovaviridae (papilloma viruses, polyoma viruses),Adenoviridae (adenoviruses), Herpeviridae (for example herpes simplexvirus (HSV) I and II, varicella zoster virus, pox viruses) andIridoviridae (for example African swine fever virus) and unclassifiedviruses (for example the etiologic agents of Spongiformencephalopathies, the agent of delta hepatitis, the agents of non-A,non-B hepatitis (class 1 enterally transmitted; class 2 parenterallytransmitted such as Hepatitis C); Norwalk and related viruses andastroviruses).

Examples of fungi include Aspergillus spp., Coccidoides immitis,Cryptococcus neoformans, Candida albicans and other Candida spp.,Blastomyces dermatidis, Histoplasma capsulatum, Chlamydia trachomatis,Nocardia spp., and Pneumocytis carinii.

Parasites include but are not limited to blood-borne and/or tissueparasites such as Babesia microti, Babesi divergans, Entomoebahistolytica, Giarda lamblia, Leishmania tropica, Leishmania spp.,Leishmania braziliensis, Leishmania donovdni, Plasmodium falciparum,Plasmodium malariae, Plasmodium ovale, Plasmodium vivax, Toxoplasmagondii, Trypanosoma gambiense and Trypanosoma rhodesiense (Africansleeping sickness), Trypanosoma cruzi (Chagus' disease) and Toxoplasmagondii, flat worms, and round worms.

As noted this invention preferably is preferably directed to the use ofan agonistic anti-human CD27 antibody optionally in association withanother moiety such as an agonistic anti-human CD40, OX-40, CTLA-4,4-1BB antibody or a cytokine or agent that depletes regulatory T cellsin treating proliferative diseases such as cancers. Cancer is acondition of uncontrolled growth of cells which interferes with thenormal functioning of bodily organs and systems. A subject that has acancer is a subject having objectively measurable cancer cells presentin the subjects' body. A subject at risk of developing cancer is asubject predisposed to develop a cancer, for example based on familyhistory, genetic predisposition, subject exposed to radiation or othercancer-causing agent. Cancers which migrate from their original locationand seed vital organs can eventually lead to the death of the subjectthrough the functional deterioration of the affected organ.Hematopoietic cancers, such as leukemia, are able to out-compete thenormal hematopoietic compartments in a subject thereby leading tohematopoietic failure (in the form of anemia, thrombocytopenia andneutropenia), ultimately causing death.

The compositions of the invention comprising a CD27 agonist andoptionally another moiety as mentioned above can be used to treat avariety of cancers or subjects at risk of developing cancer, e.g., bythe inclusion of a tumor-associated-antigen (TAA). This is an antigenexpressed in a tumor cell. Examples of such cancers include breast,prostate, colon, blood cancers such as leukemia, chronic lymphocyticleukemia, and the like. A tumor associated antigen can also be anantigen expressed predominantly by tumor cells but not exclusively.

Additional cancers include those already mentioned as well as basal cellcarcinoma, biliary tract cancer, bladder cancer, bone cancer, brain andcentral nervous system (CNS) cancer, cervical cancer, choriocarcinoma,colorectal cancers, connective tissue cancer, cancer of the digestivesystem, endometrial cancer, esophageal cancer, eye cancer, head and neckcancer, gastric cancer, intraepithelial neoplasm, kidney cancer, larynxcancer, liver cancer, lung cancer (small cell, large cell), lymphomaincluding Hodgkin's lymphoma and non-Hodgkin's lymphoma; melanoma;neuroblastoma; oral cavity cancer (for example lip, tongue, mouth andpharynx); ovarian cancer; pancreatic cancer; retinoblastoma;rhabdomyosarcoma; rectal cancer; cancer of the respiratory system;sarcoma; skin cancer; stomach cancer; testicular cancer; thyroid cancer;uterine cancer; cancer of the urinary system; as well as othercarcinomas and sarcomas.

The subject CD27 agonist, and compositions containing or combinationtherapies as defined previously can also be used to treat autoimmunediseases such as multiple sclerosis, rheumatoid arthritis, type 1diabetes, psoriasis or other autoimmune disorders. Other autoimmunedisease which potentially may be treated with the immune adjuvants ofthe invention include Crohn's disease and other inflammatory boweldiseases such as ulcerative colitis, systemic lupus eythematosus (SLE),autoimmune encephalomyelitis, myasthenia gravis (MG), Hashimoto'sthyroiditis, Goodpasture's syndrome, pemphigus, Graves disease,autoimmune hemolytic anemia, autoimmune thrombocytopenic purpura,scleroderma with anti-collagen antibodies, mixed connective tissuedisease, polypyositis, pernicious anemia, idiopathic Addison's disease,autoimmune associated infertility, glomerulonephritis) for examplecrescentic glomerulonephritis, proliferative glomerulonephritis),bullous pemphigoid, Sjogren's syndrome, psoriatic arthritis, insulinresistance, autoimmune diabetes mellitus (type 1 diabetes mellitus;insulin dependent diabetes mellitus), autoimmune hepatitis, autoimmunehemophilia, autoimmune lymphoproliferative syndrome (ALPS), autoimmunehepatitis, autoimmune hemophilia, autoimmune lymphoproliferativesyndrome, autoimmune uveoretinitis, and Guillain-Bare syndrome.Recently, arteriosclerosis and Alzheimer's disease have been recognizedas autoimmune diseases. Thus, in this embodiment of the invention thesubject CD27 agonist, typically an agonistic CD27 antibody may beadministered alone or in combination with a self-antigen against whichthe host elicits an unwanted immune response that contributes to tissuedestruction and the damage of normal tissues.

The subject anti-CD27 antibodies and combination therapies can also beused to treat asthma and allergic and inflammatory diseases. Asthma is adisorder of the respiratory system characterized by inflammation andnarrowing of the airways and increased reactivity of the airways toinhaled agents. Asthma is frequently although not exclusively associatedwith atopic or allergic symptoms. Allergy is acquired hypersensitivityto a substance (allergen). Allergic conditions include eczema, allergicrhinitis, or coryza, hay fever, bronchial asthma, urticaria, and foodallergies and other atopic conditions. An allergen is a substance thatcan induce an allergic or asthmatic response in a susceptible subject.There are numerous allergens including pollens, insect venoms, animaldander, dust, fungal spores, and drugs.

Examples of natural and plant allergens include proteins specific to thefollowing genera: Canine, Dermatophagoides, Felis, Ambrosia, Lotium,Cryptomeria, Alternaria, Alder, Alinus, Betula, Quercus, Olea,Artemisia, Plantago, Parietaria, Blatella, Apis, Cupressus, Juniperus,Thuya, Chamaecyparis, Periplanet, Agopyron, Secale, Triticum, Dactylis,Festuca, Poa, Avena, Holcus, Anthoxanthum, Arrhenatherum, Agrostis,Phleum, Phalaris, Paspalum, Sorghum, and Bromis.

It is understood that the subject antibodies, antibody containingcompositions, and conjugates thereof can be combined with othertherapies for treating the specific condition, e.g., infectious disease,cancer or autoimmune condition. For example in the case of cancer theinventive methods may be combined with chemotherapy or radiotherapy.

Methods of making antibodies against desired antigens are well known.However, until the present invention, agonistic CD27 mAbs have not beenreported.nor has the use thereof for immune therapy been suggested asclaimed herein.

By contrast, as noted above agonistic CD40 antibodies are known as isthe use thereof in immune therapies. Also, in FIG. 9 this applicationprovides the variable sequences for the heavy and light chain of apreferred exemplary chimeric agonistic CD40 antibody LOB 7/4 which wouldenable one skilled in the art to make this antibody by recombinantmethods. If the CD27 antibody is used in association with a CD40agonistic antibody, the effective amounts of the subject CD40 agonistand CD27 agonist can be determined empirically, or based onimmunologically effective amounts in animal models. The relative amountsare those which result in an enhanced CTL response and proliferation ofCD8+ T cells in vivo. Additional factors to be considered include theantigenicity, the formulation, the route of administration, the numberof immunizing doses to be administered, the physical condition, weight,and age of the individual, adverse effects and the like. Such factorsare well known to those skilled in the art and can be determined bythose skilled in the art (see for example Paoletti and McInnes, eds.,Vaccines, from Concept to Clinic: A Guide to the Development andClinical Testing of Vaccines for Human Use CRC Press (1999)). It isunderstood that the subject CD27 agonist can be administered alone or inconjunction with other adjuvants.

The subject agonist or combination thereof, e.g., CD40/CD27 agonisticantibody combination, can be administered locally or systemically by anymethod known in the art including but not limited to intramuscular,intravenous, intradermal, subcutaneous, intraperitoneal, intranasal,oral or other mucosal routes. Additional routes include intracranial(for example intracisternal, or intraventricular), intraorbital,ophthalmic, intracapsular, intraspinal, and topical administration. Theadjuvants and vaccine compositions of the invention can be administeredin a suitable, nontoxic pharmaceutical carrier, or can be formulated inmicrocapsules or a sustained release implant. The immunogeniccompositions of the invention can be administered multiple times, ifdesired, in order o sustain the desired cellular immune response. Theappropriate route, formulation, and immunization schedule can bedetermined by one skilled in the art.

In the methods of the invention, in some instances the antibody orantibody conjugate combination may be administered in conjunction withone or several antigens or other active agents, e.g., a cytokine orchemotherapeutic. These compositions and active agents containing may beadministered separately or in combination in any order that achieve thedesired enhancement of cellular immunity. Typically, these compositionsare administered within a short time of one another, i.e. within about aday of one another, typically several hours of one another, and moretypically within about an hour or less of each other.

In some instances, it may be beneficial to include a moiety on therecombinant antibody agonist which facilitates affinity purification.Such moieties include relatively small molecules that do not interferewith the function of the polypeptides in the conjugate. Alternatively,the tags may be removable by cleavage. Examples of such tags includepoly-histidine tags, hemagglutinin tags, maltase binding protein,lectins, glutathione-S transferase, avidin and the like. Other suitableaffinity tags include FLAG, green fluorescent protein (GFP), myc, andthe like.

The subject antibodies and antibody conjugates containing can beadministered with a physiologically acceptable carrier such asphysiological saline. The composition may also include another carrieror excipient such as buffers, such as citrate, phosphate, acetate, andbicarbonate, amino acids, urea, alcohols, ascorbic acid, phospholipids,proteins such as serum albumin, ethylenediamine tetraacetic acid, sodiumchloride or other salts, liposomes, mannitol, sorbitol, glycerol and thelike. The agents of the invention can be formulated in various ways,according to the corresponding route of administration. For example,liquid formulations can be made for ingestion or injection, gels orprocedures can be made for ingestion, inhalation, or topicalapplication. Methods for making such formulations are well known and canbe found in for example, “Remington's Pharmaceutical Sciences,” 18^(th)Ed., Mack Publishing Company, Easton Pa.

The subject agonistic antibodies can be expressed using any vectorcapable of directing its expression, for example a cell transduced withthe vector. Vectors which may be used include by way of examplebaculovirus, T7 based vectors for use in bacteria, yeast expressionvectors, mammalian expression vectors, viral expression vectors, and thelike. Viral vectors include retroviral, adenoviral, adeno-associatedvectors, herpes virus, simian virus 40, and bovine papilloma virusvectors.

Prokaryotic and eukaryotic cells that can be used to facilitateexpression of the subject agonistic antibodies include by way of examplemicrobia, plant and animal cells, e.g., prokaryotes such as Escherichiacoli, Bacillus subtilis, and the like, insect cells such as Sf21 cells,yeast cells such as Saccharomyces, Candida, Kluyveromyces,Schizzosaccharomyces, and Pichia, and mammalian cells such as COS,HEK293, CHO, BHK, NIH 3T3, HeLa, and the like. One skilled in the artcan readily select appropriate components for a particular expressionsystem, including expression vector, promoters, selectable markers, andthe like suitable for a desired cell or organism. The selection and useof various expression systems can be found for example in Ausubel etal., “Current Protocols in Molecular Biology, John Wiley and Sons, NewYork, N.Y. (1993); and Pouwels et al., Cloning Vectors: A LaboratoryManual”, 1985 Suppl. 1987). Also provided are eukaryotic cells thatcontain and express the subject DNA constructs.

As used herein, the term “antibody” is used in its broadest sense toinclude polyclonal and monoclonal antibodies, as well as antigen bindingfragments thereof. This includes Fab, F(ab′)2, minibodies, single chainAb and Fv fragments.

In addition the term “antibody” includes naturally antibodies as well asnon-naturally occurring antibodies such as single chain antibodies,chimeric antibodies, bifunctional and humanized antibodies. Preferredfor use in the invention are chimeric, humanized and fully humanantibodies. Methods for synthesis of chimeric, humanized, CDR-grafted,single chain and bifunctional antibodies are well known to those skilledin the art. In addition, as noted antibodies specific to CD27 are knownand available and can be made by immunization of a suitable host with aCD27 antigen, preferably human CD27 antigen. As noted in the presentinvention if a CD40 antibody is used the CD40 antibody may comprisechimeric LOB 7/4 having the variable heavy and light chain sequencescontained in FIG. 9 the synthesis of which is depicted schematically inFIGS. 10 and 11.

The agonistic CD27 antibodies of the present invention may be intact orengineered For example, the CD27 mAb may be fully or partiallyglycosylated and/or selected for increased or diminished binding tohuman effector systems such as complement, FcR-bearing effectors, suchas macrophages, or to extend or reduce half-life. These modificationscan be made to improve effectiveness and potentially also reduce toxicside effects.

It is understood that modifications which do not substantially affectthe activity of the various embodiments of this invention are alsoprovided within the definition of the invention provided herein.Accordingly, the following examples are intended to illustrate but notlimit the present invention.

EXAMPLES

Materials and Methods

(The following materials and methods were used in Examples 1-6.)

Animals and cell lines BALB/c and C57Bl/6 mice were supplied by Harlan(Blackthorn, Oxon, U.K.) and maintained in local animal facilities. TheBCL1 (Slavin and Strober 1978) mouse B lymphoma line was maintained byin vivo by i.p. passage in BALB/c mice. Spleens were removed at theterminal stage of disease and single cell suspensions were prepared asdescribed previously. P BCL1 cells are a subline derived from BCL1 andgrow in culture (Midge, Honeychurch et al. 2000). Animal experimentswere conducted under license according to the U.K. Home Office licenseguidelines and approved by the University of Southampton EthicalCommittee.

Antibodies and reagents The anti-CD40 treatment mAb used in this studywas 3/23 (originally provided by G. Klaus, National Institute of MedicalResearch, London, U.K.). The anti-4-1BBL mAb, AT113-2, and the anti-CD7mAb TAN1-6 (Taraban, Rowley et al. 2004) were raised in house byimmunizing rats with 4-1BBL-Fc fusion protein or soluble recombinant CD7protein respectively. For the preparation of mAb, hybridoma cells wereexpanded in stationary culture and IgG prepared from supernatant usingProtein G-coupled Sepharose beads. Antibodies used for flow cytometricanalysis were: ID3 (anti-CD19) (Krop, de Fougerolles et al. 1996)16-10A1 (anti-B7-1) and GL-1 (anti-B7-2) (both American Type CultureCollection), ICAM-1 (ref), N418 (anti-CD11c) (ATCC), Mc10-6A5 (anti-BCL1Id mAb) (George, McBride et al. 1991), LOB12/3 (anti-4-1BB) (Taraban,Rowley et al. 2004), YTS 169 (anti-CD8), all prepared and PE- orFITC-labeled in house; PE-labeled anti-CD27 and APC-labeled anti-CD8(both Pharmingen). Soluble fusion proteins, sCD70-Fc and s4-1BBL-Fc,were generated as described previously (Rowley and Al-Shamkhani 2004).

BIACORE Analysis Biacore analysis of the binding of TAN1-6 and AT113-2was performed as described previously (Al-Shamkhani, Mallett et al.1997).

Adoptive Transfer of OT-1 cells OVA-specific H-2Kb-restricted TCRtransgenic T cells (1×106) from OT-I mice were injected i.v. intosex-matched C57BL/6 recipients. After 1 or 2 days, T cells were primedby i.p. administration of OVA (5 mg) in combination with: anti-CD40 orcontrol anti-A31 lymphoma Id mAb (500 μg of each), or anti-CD40 andanti-CD70/anti-4-1BBL mAb (500 μg of each). The next day these micereceived an additional injection of control anti-A31 lymphoma Id mAb,anti-CD70 or anti-4-1BBL mAb (500 μg). For tracking Ag-specific T cells,blood samples (50 μl), were stained with PE-labeled H-2Kb OVAp tetramers(Proimmune or Beckman Coulter) and APC-labeled anti-CD8□ (Pharmingen)(Taraban, Rowley et al. 2004).

Monitoring of endogenous anti-OVA response C57BL/6 mice were primed byi.p. administration of OVA (5 mg) in combination with anti-CD40 andeither control (anti-A31 lymphoma Id), anti-CD70 or anti-CD137L mAb (500μg of each). The next day mice received an additional injection ofeither control, anti-CD70 or anti-CD137L mAb (500 μg). For trackingAg-specific T cells, 6 days after priming, blood samples (50 μl), werestained with PE-labeled H-2Kb OVAp tetramers and APC-labeled anti-CD80)(Pharmingen), and flow cytometric analysis was performed using aFACSCalibur (BD Biosciences, Mountain View, Calif.).

Flow Cytometric Analysis of Splenic Lymphocytes and DCs Following Tumorand Anti-CD40 mAb

Age-matched BALB/c mice were given 5×107 BCL1 cells on day 0 (i.v.) andthen 1 mg of anti-CD40 mAb or isotype-matched control (i.v.) when thelevel of tumor cells in the spleen had reached approximately 5% of totalcells, typically day 4 post-tumor. In experiments to look at the effectof anti-4-1BBL and anti-CD70 mAb on the response of anti-CD40 mAbtreatment, 0.5 mg of the blocking mAb was injected i.p. 4 h prior to theinjection of anti-CD40, and then again on days 1 and 3 after theanti-CD40 mAb. Animals were sacrificed on the days indicated, spleensremoved and suspensions prepared. BCL1 tumor cells were detected usingPE-anti-CD19 and FITC-anti-BCL1 Id. Changes in number and phenotype ofCD8+ lymphocytes were followed using APC-anti-CD8a and PE-anti-4-1BB and-anti-CD27. Flow cytometric analysis was performed using a FACSCalibur(BD Biosciences).

For DC analysis, whole spleens were coarsely chopped and digested in 5ml of 1 mg/ml collagenase D (Roche) and 0.05 mg/ml DNaseI (Sigma) inRPMI-1640 and gently agitated for 30 min at 37 degrees C. 20 ml ofmedium was then added and a single cell suspension prepared. The cellswere washed once, resuspended, and samples labeled for flow cytometryusing PE-anti-CD11c and FITC-anti-B7.1, B7.1, ICAM, 4-1BB, 4-1BBL, andCD70 in the presence of the anti-FcgII and III receptor mAb, 2.4G2.7-aminoactinomycin-D (7AAD) at a final concentration of 2 μg/ml wasadded to the samples 15 minutes before analysis so that dead andautofluorescent cells could be gated out using FL3. Flow cytometricanalysis was performed using a FACSCalibur.

Immunotherapy Groups of 5 age-matched mice were injected i.v. with 107BCL1 cells on day 0 and then with anti-CD40 i.v. on days 4 to 7 (250mg/day) and blocking mAb i.p. on days 4, 7, 9 and 11 (500 mg/day) whereindicated.

In vivo killing assay Mice were injected with 2×107 BCL1 cells i.p., and48 hours later with 1 mg anti-CD40 i.p. After 8 days, groups of threemice were injected i.p. with 1 mg of the appropriate blocking mAb andthen 5 hours later with 2×107 CFSE labeled splenocytes from terminalBCL1 tumor-bearing mice. Twenty four hours later, cells were harvestedfrom the peritoneal cavity by washing and staining with PE-labeledanti-BCL1 Id and APC-labeled anti-CD8.

Cytotoxicity assay A standard 4-h 51Cr release assay was used to assesscytotoxic activity of splenic effectors as described previously (Tutt,O'Brien et al. 2002). Briefly, splenic homogenates were prepared fromBCL1-bearing mice 4-5 days after anti-CD40 treatment. Remaining tumorcells were removed using FITC-anti-Id mAb, followed by anti-FITC MACSbeads and LS columns (Miltenyi Biotec, Bergisch Gladbach, Germany). Theremaining cells were used as effectors. PBCL1 cells were labeled with51Cr and used as targets. To assess the effect of mAb on cytotoxicity,they were included at a final concentration of 50 μg/ml. The maximumrelease of radioactivity was calculated using target cells to which 150μl of 1% Nonidet P-40 had been added. The percentage of specific 51Crrelease was calculated using the standard formula: percentage ofspecific release=[(sample release−background release)/(maximumrelease−background release)]×100.

Example 1: TAN1-6 (ANTI-CD70) and AT113-2 (Anti-4-1BBL) Block the4-1BBL/4-1BB and CD70/CD27 Interactions Respectively In Vitro and InVivo

In this experiment contained in FIG. 1, in both 1 a and 1 b BALB/csplenocytes were activated for 48 hours with 1 microgram/ml of anti-CD3to up-regulate the expression of CD70 and 4-1BB on T cells. In 3 a, thebinding of sCD70-IgFc fusion protein (1.125 micrograms/ml) to theactivated splenocytes was detected using the FITC-labeled anti-human FcmAb (SB2H2, 10 micrograms/ml) in the presence of s4-1BBL-Ig Fc fusionprotein (1.125 micrograms/ml) to the cells was detected using theFITC-labeled anti-human Fc mAb SB2H2 in the presence of TAN1-6 (solidline) or AT113-2 (dashed line) (50 micrograms/ml). In both a and 1 b thefilled histograms show a control of hIgG detected with SB2H2. In 1 c,the endogenous Ova-specific CD8 T-cell response in C57Bl/6 mice wasmonitored following i.p. administration of OVA (5 mg) and anti-CD40 (1mg) in combination with either control IgG (anti-A31 idiotype),anti-CD70 (TAN1-6) or anti-4-1BBL (AT113-2) mAb (0.5 mg of each). After24 hours, the mice received repeated injection of control, anti-CD70 oranti-4-1BBL mAb. Six days later, the OVA-specific T cells in theperipheral blood were detected with APC-labeled anti-CD8 and PE-labeledH-2 Kb SIINFEKL tetramer.

Example 2: Effect of Anti-4-1BBL (AT113-2) and Anti-CD70 (TAN1-6) on theTherapeutic Activity of Anti-CD40 in BCL1 Mice

In this experiment shown in FIG. 2, groups of 5 mice were inoculated ivwith 1×107 BCL1 tumor cells o day 0, and treated iv with anti-CD40 ondays 4, 5, 6 and 7 post-tumor (500 micrograms per day). Where shown, thegroups also received anti-4-1BBL and/or anti-CD70 on days 4, 7, 9 and 11(500 micrograms/mAb/day, i.p. Mice were monitored for tumor development.These results in FIG. 4 represent and contain the results of one of 3similar experiments.

Example 3: Effect of Anti-4-1BBL and Anti-CD70 on the Accumulation ofCD8+ T Cells and The Eradication of BCL1 Tumors After Anti-CD40 mAbTherapy

In this experiment the results of which are contained in FIG. 3, groupsof mice were inoculated with 5×107 BCL1 cells iv and four days later(day 0 in the figure) treated with anti-CD40 mAb, at which point thelevel of Id+-tumor cells in the spleen was between 3 and 5%. Whereindicated, the mice also received anti-4-1BBL and/or anti-CD70 mAb ondays 0, 1, and 2 (500 micrograms/mAb/day). On the days indicated,splenic tumor and CD8+ T cells were monitored by flow cytometry asdescribed in the Materials and Methods herein. The experiment in FIG. 3ashows the total number of splenic tumor cells and 3 b the total numbersof CD8+ cells after treatment of BCL1. The points are the mean ofduplicate animals, and the results shown represent the results of 4similar experiments.

Example 4: Anti-4-1BBL (AT113-2) and Anti-CD70 (TAN1-6) Do Not Affectthe Anti-CD40 Induced-Phenotype Changes or Changes in the Number ofSplenic DCs from BCL1 Mice

In the experiment in FIG. 4 mice were inoculated with 5×107 BCL1 cellsiv and four days later (day 0 in the figure) treated with anti-CD40 (1mg) mAb. Where indicated, the mice also received anti-4-1BBL oranti-CD70 i.p four hours prior to anti-CD40 treatment, and again 1 and 2days later (500 micrograms/day). On day three, the total number ofsplenic DCs were analyzed as in FIG. 2. The results in FIG. 4 representthe means of duplicate mice and contain the results of 1 of 2 similarexperiments.

Example 5: Anti-4-1BBL and Anti-CD70 Do Not Inhibit the Effector Stageof the Response In Vivo or In Vitro

This experiment is contained in FIG. 5. In FIG. 5a mice were given .i.p.injections of 2×107 BCL1 cells and 48 hours later anti-CD40 mAb (1 mg).After an additional 8 days, groups of three mice were injected i.p. with1 mg of the appropriate blocking antibody and then 5 hours later with2×107 CFSE labeled splenocytes from BCL1 tumor-bearing mice. 24 hourslater, cells were harvested from the peritoneal cavity, stained withPE-labeled anti-BCL1 Id, and evaluated by flow cytometry. The gated,double positive (CFSE+ and Id+) cells represent the surviving tumorcells. In 5 b) mice were inoculated with 5×107 BCL1 i.v. and treated 4days late with anti-CD40 mAb (1 mg, i.v.). 5 days after anti-CD40,splenic lymphocytes were prepared and the remaining tumor cells, removedusing PE-labeled anti-BCL1 Id mAb and anti-PE beads as describe in theMaterial and Methods. The remaining splenocytes (approximately 40% CD8+)were used as effectors, and incubated with 51 Cr-labeled IIBCL1 cells astargets, alone, or in the presence of anti-CD40, anti-CD8, anti-4-1BBL,or anti-CD70 at 100 micrograms/ml. The results therein are expressed asthe percentage specific lysis of target cells.

Example 6: Therapeutic Potency of Anti-CD27 Against A31 and BCL1Lymphoma

In this experiment shown in FIG. 6, mice were given injections of 2×107BCL1 cells iv and then later treated iv with Control IgG, anti-CD40 mAb,anti-CD27 or anti-4-1BB mAb (1 mg). The mice were monitored twice dailyand the results in the figure represent one of two similar experiments.Particularly, one day after adoptive transfers of 4×106 CD8+ OT-Ilymphocytes (day 0), the recipient B6 mice were immunized iv with 30mmol OVA peptide (SINFEKL, OVA 257-264). The animals also received fouriv injections (on days 90, 1, 2 and 3; 250 micrograms per injection) ofrecombinant soluble CD70 or anti-mouse CD27 (preparation thereofdescribed in the next example) or control mAb. For tracking ofOVA-specific CD8+ T cells in vivo, blood samples were stained with PEH-2KB OVA 257-264 tetramer and APC anti-CD8alpha. Expansion ofOVA-specific CD8+ T cells is shown on day 4, at the peak of theresponse.

Example 7: Preparation of Anti-Mouse CD27 mAb AT124-1

Mouse CD27-huFc secreting CHO cells were prepared and supernatanttherefrom processed over Protein A to purify the fusion protein. Lourats were immunized with 50 micrograms/dose fusion protein in CFA, IFCA,then PBS following standard methods. Hybridomas were screened initiallyon the fusion protein and subsequently on the cells. The cells were ConA activated spleen cells, i.e., activated T cells.

Compared to commercial Hamster CD27 (LG3A10 (Becton Dickinson) P 04 2115), AT124-1 (inventive antibody) was partially or was not blocked byCD7O-Fc (110 down to 80 MFI) whereas LG3A10-PE was blocked 1500 down to180 MFI. This experiment was repeated with P 04 2 130 with similarresults.

As shown in FIGS. 8A and 8B both the inventive rat anti-mouse CD27(AT124-1) and the positive control hamster anti-mouse CD27 (LG3A10)labeled the mouse CD27 transfected cells (FIGS. 8A and 8B,respectively). Neither antibody bound untransfected cells.

Results

Phenotypic Changes in Splenic CD8 T Cells and DC During Anti-CD40Treatment of BCL1 Lymphoma

Effect of Blocking 4-1BBL/4-1BB and CD70/CD27 Interactions on theTherapeutic Response to Anti-CD40

The inventors investigated the importance of the 4-1BB and CD27molecules to the therapeutic response seen following CD40 mAb treatmentof tumor-bearing mice. The effect of disrupting 4-1BBL/4-1BB andCD70/CD27 interactions was investigated using blocking mAb specific for4-1BBL and CD70. That AT113-2 (anti-4-1BBL) and TAN1-6 (anti-CD70) wereable to disrupt 4-1BBL/4-1BB, and CD70/CD27 interactions respectively,was confirmed by showing that they blocked the binding of s4-1BBL-Fc andsCD70-Fc to activated T cells (FIGS. 1a and b ). Furthermore, both mAbbound to their target antigens with similar affinity as determined byBIACore analysis. The KD values for AT113-2 (anti-4-1BBL) and TAN1-6(anti-CD70) were 3.8×10-9 and 3.0×10-9 respectively. To confirm that theanti-CD70 and anti-4-1BBL mAb were able to block responses in vivo, theinventors investigated their effect on the endogenous CD8 response toOVA in C57BL/6 mice (FIG. 1c ). As shown previously (Taraban, Rowley etal. 2004), the CD40 mediated expansion of endogenous OVA-specific CD8 Tcells was almost completely inhibited by anti-CD70 mAb. In comparison,the anti-4-1BBL mAb resulted in a 50% reduction in the level ofanti-CD40 induced OVA-specific T cells.

The inventors then determined the effect of the anti-CD70 andanti-4-1BBL mAb on the therapeutic response of BCL1 tumor bearing miceto anti-CD40 mAb. Mice were inoculated with 1×107 BCL1 tumor cells i.v.on day 0, and treated with anti-CD40 on days 4, 5, 6 and 7 post tumor.FIG. 2 shows that, as we have previously reported (French, Chan et al.1999; Tutt, O'Brien et al. 2002) BCL1-bearing mice treated withanti-CD40 are protected against lymphoma and go on to become long-termsurvivors (survived more than 100 days). However, CD70 mAb completelyblocked the therapeutic activity of the CD40 mAb treatment. In contrast,mice receiving the blocking 4-1BBL mAb survived for a median of 73 days,compared with 18 days for the untreated group, and then succumbed todisease, suggesting some impairment in the development of long-termimmunity in the absence of 4-1BB co-stimulation. The inventorsconsistently found that CD40 mAb-treated mice receiving both anti-4-1BBLand anti-CD70 succumbed to tumor slightly earlier than control untreatedanimals (median survival 14 days compared with 17 days), suggesting thatthis combination might block the CD40 mAb therapeutic activity and alsoperhaps a weak spontaneous response to the tumor.

The inventors next investigated the effects of blocking with anti-4-1BBLand anti-CD70 mAb on the kinetics of the CD8 response to CD40 mAbtreatment. FIG. 3 shows the effects of the 4-1BBL and anti-CD70 mAb onthe growth of BCL1 (FIG. 3a ) and the CD8 T-cell response (FIG. 3b ) inthe spleen of CD40 mAb-treated mice. Compared to CD40 mAb alone,treatment with CD40 mAb in the presence of anti-4-1BBL resulted in adelay in the expansion of CD8 T-cell of about 2 days resulting in theslower clearance of tumor cells from the spleen. However, althoughdelayed, the final number of CD8 cells was at least as high as withanti-CD40 alone. Interestingly, in these experiments, where the initialtumor dose was 5×107 cells, rather than the 1×107 cells used in mostimmunotherapy experiments, mice treated with CD40 mAb and blocking4-1BBL mAb went on to become long-term survivors and were immune tore-challenge with tumor (results not shown).

In contrast, anti-CD70 mAb completely blocked the anti-CD40-inducederadication of tumor cells. This lack of response was reflected in thefailure of the CD8+ T cell population to expand and explains the abilityof CD70 mAb to completely block the CD40 mAb-mediated therapy of BCL1tumor shown in FIG. 2. The results demonstrate that the blocking ofanti-CD70/anti-CD27 interactions has a profound effect on thetherapeutic response. By contrast, the influence of the 4-1BBL/4-1BBinteraction appears relatively modest.

Since it was observed that both anti-4-1BBL and anti-CD70 had an effecton the CD40 mAb-induced CD8 response (not shown) the inventors furtherassessed whether they were having any effect on the anti-CD40 inducedchanges in DC number and phenotype in tumor-bearing mice. As shown inFIG. 4, neither anti-4-1BBL nor anti-CD70 inhibited the increase inexpression of B7.1, B7.2 and ICAM (a) or in the number of DC recovered(b) after anti-CD40 treatment of tumor-bearing mice. Thus blocking ofthese co-receptors does not influence DC recruitment and is unlikely toaffect their activation.

CD27 and 4-1BB do not operate at the effector stage of the CD40mAb-mediated anti-tumor response Since we have observed that the BCL1tumor cells themselves express both CD70 and 4-1BBL (FIG. 5a ), and alsosince it has been reported that CD70 is also expressed by activated Tcells (very recently by Steve Rosenberg), the inventors theninvestigated whether the anti-4-1BBL and anti-CD70 had an effect on theeffector stage of the CTL response. It was observed (unpublishedobservations) that when BCL1 tumor cells are administered and treatedwith anti-CD40 mAb in the peritoneal cavity, then the CD8 response inthat compartment peaks at day 8 post treatment, at which pointapproximately 30% of the recovered lymphocytes are CD8+ (5% in naïvemice), and clears the tumor rapidly. This system was used to investigatethe effector stage of CTL-mediated killing in vivo. Mice received i.p.BCL1 and anti-CD40 mAb as described and then 8 days after anti-CD40 wereinjected i.p with 1 mg of anti-CD8, anti-CD4, anti-CD70 or anti-4-1BBLblocking mAb, or control PBS. Five hours later they were injected i.p.with 2×107 CFSE-labeled splenocytes from terminal BCL1-bearing mice asCTL targets. Labeled splenocytes were also injected into naïve mice as acomparison. Twenty four hours later, peritoneal cavity cells wereharvested, and analyzed for the presence of CFSE labeled BCL1 tumorcells. The peritoneal cavity cells harvested from naïve mice included22-27% CFSE-labeled tumor cells (FIG. 5). In contrast, tumor cells werecompletely eradicated in mice that had undergone anti-CD40 treatment ofBCL1., underlining the effectiveness of this treatment. As expected,injection of anti-CD8 mAb blocked the clearance of CFSE-labeled tumorcells from the peritoneal cavity. However, anti-CD4, anti-4-1BBL andanti-CD70 had no effect on the clearance of transferred tumor cells.These results were confirmed in a cytotoxicity assay using splenocytestaken from BCL1-bearing mice 5 days after anti-CD40 treatment, afterremoval of residual tumor cells, as effectors, and the results are shownin FIG. 5c . Neither anti-4-1BBL nor anti-CD70 had a direct effect onthe cytotoxicity of immune splenocytes.

Direct immunotherapy via 4-1BB and CD27.

The results above suggest that the striking CTL response seen aftertumor inoculation and CD40 mAb treatment, is partially dependent ontriggering via 4-1BB and completely dependent on interactions betweenCD27 and CD70. Therefore, to investigate if we could short-cut theagonistic activity via CD40 and simulate these co-receptors directly, wenext investigate the therapeutic potency of agonistic anti-4-1BB oranti-CD27 against the BCL1 lymphoma. As shown in FIG. 6, the anti-CD70mAb was completely protective for BCL1 tumor and gave results which werevery similar to those obtained with anti-CD40 mAb. In contrast theanti-4-1BB mAb had a modest therapeutic activity for this tumor, givinglittle prolongation of survival.

Agonistic CD40 mAb is one of the most exciting reagents currently underinvestigation for human use. For some time it has been known that, evenas a monotherapy, anti-CD40 mAb has efficacy in a range of vaccinesettings, including induction of tumor immunity in murine models andmore recently in patients. For example, recent data from Vonderheide etal (2006) have shown that a fully human CD40 mAb can deliver PR in 27%of melanoma patients when given at close to its MTD (0.2 mg/kg).

While it is known that CD40 plays a critical role in the immune system,particularly in the activation of APC, such as DC, much is unknown aboutthe role of co-receptor:ligand interaction between the activated DC andresponding CTL. A wealth of evidence shows that triggering via CD40,such as that delivered with a mAb, activates the DC resulting inupregulation of MHC, B7.1/2, and various co-receptors and cytokines, toallow generation of effector CTL. Furthermore, it is generally foundthat using the appropriate CD40 agonist replaces the need for T-helpercells, and given the paucity of helper epitopes expressed by certaintumors, perhaps this explains the success of CD40 agonists in a cancersetting. In the subject current invention we have investigated the roleof 4-1BBL and CD70, and shown how important signals via their respectivereceptors, 4-1BB and CD27, are in controlling CTL responses.Additionally, the inventors found that CD40 treatment promotes marked DCexpansion and, as has been widely reported, an increase in theexpression of I-CAM-1, B7.1 and B7.2 on splenic DC, consistent withtheir activation or ‘licensing’ for antigen presentation. It isgenerally accepted that the CD40 mAb-induced anti-tumor response is dueto cross-presentation of tumor antigens by activated APCs (van Mierlo,Boonman et al. 2004). Consistent with this, we have previously shownthat CD40 mAb therapy is at least partially effective with CD40-negativetumors (French, Chan et al. 1999). Splenic DC from mice receiving tumoralone showed a degree of activation, with partial up-regulation of B7.1,B7.2 and ICAM-1. However, somewhat surprisingly in view of the cleareffect of both anti-CD70 and anti-4-1BBL on the CD40-induced response tolymphoma (FIG. 2), in spite of numerous attempts, only small andtransient increases in the expression of CD70 and 4-1BBL were detectedafter anti-CD40 treatment, and even these were abrogated in the presenceof tumor. Interestingly, 4-1BBL expression on in vitro activated DCs hasalso been shown to be low (DeBenedette, Shahinian et al. 1997; Futagawa,Akiba et al. 2002), although Diehl et al (Diehl, van Mierlo et al. 2002)have shown strong expression on splenocytes in response to anti-CD40 invivo.

The low expression of 4-1BBL and CD70 on splenic DC in vivo, may reflecta transient expression of these two ligands or continual modulation byengagement of their receptors 4-1BB and CD27. Interestingly, we findthat treatment of BMDC in vitro does promote CD70 expression, perhapsbecause it is not encountering CD27. While CD70 expression has beendemonstrated on in vitro activated DCs (Futagawa, Akiba et al. 2002;Tesselaar, Xiao et al. 2003) (Taraban, Rowley et al. 2002; Bullock andYagita 2005), in vivo expression of CD70 on DCs has generally been lowand transient (Tesselaar, Xiao et al. 2003; Hendriks, Xiao et al. 2005),presumably to avoid inappropriate activation of T cells via theconstitutively expressed CD27 (Tesselaar, Arens et al. 2003).

Despite this lack of CD70 expression, mAb against 4-1BBL or CD70 wereeffective in blocking the anti-tumor response and in the latter case theinhibition was complete. Such blocking appeared to operate at the levelof CTL stimulation, since while these blocking mAb prevented thegeneration of cytotoxic T cells, they had not effect on the number oractivation of splenic DC, or on the cytotoxic function of the CD8 Tcells once they had been generated. Together these studies show that4-1BB and particularly CD27 are critical in controlling CTL responsesduring anti-CD40 mAb treatment, most likely to act at the level of theinteraction between the DC and the responding CD8 T cells.

The expression of 4-1BB on CD8 T cells during the development of theCD40-induced anti-lymphoma response was consistent with the establishedpattern (Vinay and Kwon 1998; Takahashi, Mittler et al. 1999) with 4-1BBrapidly induced on the expanding CD40-induced splenic CTL population(FIG. 1), and then lost concomitant with the eradication of tumor cells,but before the contraction of the CTL population one to two days later(Tutt, O'Brien et al. 2002). The sustained expression of 4-1BB over 4days during the response, presumably because of continuous antigenavailability, resembles that found during persistent infection and graftrejection (Tan, Ha et al. 2000; Seo, Park et al. 2003). In untreatedmice in the terminal stage of disease, the inventors identified a clearpopulation of 4-1BB+CD8 T-cells, supporting the idea that BCL1 alone isable to evoke a weak, ineffectual T-cell response, which is then boostedto an effective level by treatment with CD40 mAb.

While 4-1BB is induced on T cells and there is evidence that it providesco-stimulation for T-cell proliferation (20), it is believed to impactprimarily on cell survival (Takahashi, Mittler et al. 1999; Lee, Park etal. 2002), by inducing the expression of anti-apoptotic proteins (Lee,Park et al. 2002). Although the importance of 4-1BB in the developmentof CTL responses has been demonstrated in the allograft situation, whereblockade of 4-1BB/4-1BBL interactions has been shown to result in theenhanced graft survival, with inhibition of the expansion ofalloreactive T cells and reduced CTL activity (Cho, Kwon et al. 2004),studies in 4-1BB−/− and 4-1BBL−/− mice have shown only relatively minorimpairment in primary CTL responses to virus infections (Tan, Whitmireet al. 2000; Bertram, Dawicki et al. 2004), with a more pronouncedeffect on the recall response after rechallenge (Dawicki and Watts2004). Importantly, studies using agonistic anti-4-1BB mAb in vivo haveshown that they are able to promote effective therapy in a range ofpoorly immunogenic tumor models (Wilcox, Flies et al. 2002) and that4-1BB signaling is important predominantly during CD8 responses(Shuford, Klussman et al. 1997).

In contrast to 4-1BB, CD27 is constitutively expressed on naive T cells(Gravestein, Blom et al. 1993). Following anti-CD40 treatment of tumor,we were able to detect CD27-high and -low populations (FIG. 1c ) in theexpanding splenic CD8+ cells, possibly representing primed cells with atransient up-regulation of CD27 (Gravestein, Blom et al. 1993; Lens, deJong et al. 1996) and a population of senescent terminal effectors asdescribed by Kaech et al (Kaech, Tan et al. 2003) respectively. Theexpression of its ligand, CD70, on DC is tightly regulated, thuspreventing inappropriate activation of T cells (Tesselaar, Arens et al.2003). However, recently, constitutive triggering of CD27 by CD70 hasbeen shown to enhance both the expansion and the activity of CD8+ cellsin response to viral or tumor challenge (Arens, Schepers et al. 2004).We have recently shown that soluble CD70 promotes strong primary andsecondary CTL responses in vivo (Rowley and Al-Shamkhani 2004).Conversely, CD27-deficient mice develop impaired CD8+ responses toinfluenza virus, and, like 4-1BB, CD27 appears to promote the survivalof activated CD8+ T cells during the primary response (Hendriks,Gravestein et al. 2000; Hendriks, Xiao et al. 2003), but that, unlike4-1BB, it is also important in CD4+ responses. The CD27-deficient micealso show an impaired response to rechallenge (Hendriks, Gravestein etal. 2000).

In addition to the well established idea that T cell expansion requiresco-stimulation between ligands on DC and co-stimulatory receptors on Tcells, the detection of these molecules on other cell types suggests arole for a more complex network of interactions during the T cellresponse. For example, CD70 has been detected on activated T cells(Borst, Hendriks et al. 2005; Hendriks, Xiao et al. 2005) and 4-1BB onactivated DC both in vivo and in vitro. Although the inventors could notdetect CD70 on activated T cell in our study, 4-1 BB was clearlydetected on activated DC during the course of the response (results notshown).

Finally, we confirmed was the relative importance of triggering via CD70using an agonistic CD27 mAb as therapeutic agents for mouse lymphoma.The data herein show that CD27 mAb was as effective as CD40 mAb indeveloping tumor immunity. Ongoing work is investigating the mechanismof action of this mAb, which presumably works by stimulating the T cellsdirectly. The total dependency of the anti-CD40 induced CD8 T-cellresponse in both the OVA and the BCL1 lymphoma models on CD70/CD27interactions is consistent with the reported effects of the CD70:CD27co-stimulatory pathway in studies using viral models. While theimportance of 4-1BB/4-1BBL interactions in the development of CTLresponses has been demonstrated in the allograft situation, whereblockade of 4-1BB/4-1BBL interactions results in to enhanced graftsurvival (DeBenedette, Wen et al. 1999; Cho, Kwon et al. 2004), studiesin 4-1BB−/− and 4-1BBL−/− mice have shown only relatively minorimpairments in primary CTL responses to LCMV and influenza infections(Bertram, Dawicki et al. 2004) (Tan, Whitmire et al. 1999) (Hendriks,Xiao et al. 2005). The observation that 4-1BBL−/− mice showed a moresevere defect in their response after immunization with a lipidated LCMVpeptide (Tan, Whitmire et al. 2000) suggested that 4-1BB-co-stimulationis more critical where antigenic stimulation is weak or limiting. Incontrast, CD27-deficient mice develop impaired CD8+ responses toinfluenza virus in the lung and draining lymph node, although,interestingly, anti-viral CD8 cell expansion in the spleen wasrelatively unaffected (Hendriks, Xiao et al. 2003) in this system. Both4-1BB and CD27-deficient mice have been shown to demonstrate an impairedsecondary response to viral rechallenge (Hendriks, Gravestein et al.2000) (Dawicki and Watts 2004). While our experiments showed that thepresence of anti-4-1BBL mAb did not prevent initial clearance of thetumor after anti-CD40 treatment, in our immunotherapy experiments,animals receiving anti-CD40 and anti-4-1BBL mAb did not go on to becomelong term survivors (FIG. 2), suggesting some impairment in thedevelopment of their long-term immunity. In summary, these results andthe underlying conclusions based thereon enumerated supra suggest thatmono- and combination therapies using agonistic CD27 antibodies, e.g.,synergistic agonist combinations will provide a novel means of promotingTh1 immunity and CD8+ T cell proliferation and may be used in thetreatment of cancer, infection, inflammation, allergy and autoimmunityand for promoting the efficacy of vaccines.

It is to be understood that the invention is not limited to theembodiments listed hereinabove and the right is reserved to theillustrated embodiments and all modifications coming within the scope ofthe following claims.

The various references to journals, patents, and other publicationswhich are cited herein comprise the state of the art and areincorporated by reference as though fully set forth.

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The invention claimed is:
 1. A method for treating cancer comprisingadministering to a subject in need thereof an additively orsynergistically effective amount of: (i) at least one CD27 agonist whichcomprises an agonistic anti-human CD27 antibody, or an agonistic antigenbinding fragment thereof elicits an antitumor immune response in vivo bypromoting the proliferation of tumor-associated antigen-specific T cellsand (ii) at least one other immune agonist selected from a CD40 agonist,an agonistic 4-IBB (CD137) antibody, an agonistic OX-40 antibody, anagonistic CD28 antibody, an agonistic CTLA-4 antibody, a moiety thatdepletes regulatory T cells, a cytokine or a TLR agonist wherein saidagonists may be administered in combination or separately, in eitherorder, under dosing conditions whereby these agonists elicit an additiveor synergistic effect on antitumor immunity compared to the separateadministration of either agonist.
 2. The method of claim 1, wherein saidCD27 agonist is an agonistic anti-human CD27 antibody.
 3. The method ofclaim 2 wherein said antibody comprises a human constant region.
 4. Themethod of claim 2 wherein said antibody comprises a human IgG1 or IgG3constant region.
 5. The method of claim 1, wherein said CD27 agonist isan agonistic anti-human CD27 antibody or agonistic anti-human CD27antibody fragment which recognizes an extracellular portion of humanCD27.
 6. The method of claim 1 wherein the agonist of (ii) comprises a4-IBB agonist.
 7. The method of claim 6 wherein the agonist of (ii)comprises a 4-IBB agonist antibody.
 8. The method of claim 1 wherein theagonist of (ii) comprises a CD40 agonistic antibody or a soluble CD40Lfusion protein or soluble CD40 fragment or a conjugate thereof.
 9. Themethod of claim 8 wherein the CD40 agonist antibody is a chimeric, humanor humanized antibody that specifically binds human CD40.
 10. The methodof claim 9 wherein said anti-CD40 antibody comprises SC26, CP-870,893,or LOB 7/4 wherein LOB 7/4 is a human IgG2 antibody possessing theidentical heavy and light chain variable regions recited in FIG.
 9. 11.The method of claim 1 wherein the agonistic anti-CD27 antibody is achimeric, human or humanized antibody or is a chimeric, human orhumanized anti-CD27 antibody fragment.
 12. The method of claim 1 whereinsaid CD27 agonist and said other immune agonist are administered underdosing conditions whereby these agonists elicit an additive orsynergistic effect on the proliferation of tumor-associatedantigen-specific T cells compared to the proliferation oftumor-associated antigen-specific T cells when either agonist isadministered alone.